Glycobiology Advance Access originally published online on May 25, 2005
Glycobiology 2005 15(10):943-951; doi:10.1093/glycob/cwi082
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Characterization of a novel galactose ß1,3-N-acetylglucosaminyltransferase (ß3Gn-T8): the complex formation of ß3Gn-T2 and ß3Gn-T8 enhances enzymatic activity
2 Department of Biochemistry, Sasaki Institute, 2-2, Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062, Japan; and 3 CREST, Japan Science and Technology Agency, 4-1-8 Honcho Kawaguchi, Saitama, Japan
1 To whom correspondence should be addressed; e-mail: yamashita{at}sasaki.or.jp
Received on February 23, 2005; revised on May 18, 2005; accepted on May 19, 2005
We characterized a novel member of the ß1,3-N-acetylglucosaminyltransferase (ß3Gn-T) gene family, ß3Gn-T8. A recombinant soluble form of ß3Gn-T8 was expressed in Pichia pastoris (P. pastoris), and its substrate specificity was compared with that of ß3Gn-T2. The two enzymes had similar substrate specificities and recognized tetraantennary N-glycans and 2,6-branched triantennary glycans in preference to 2,4-branched triantennary glycans, biantennary glycans, and lacto-N-neotetraose (LNnT), indicating their specificity for 2,6-branched structures such as [Galß1
4GlcNAcß12(Galß14GlcNAcß16)Man
1 6Man]. Interestingly, when soluble recombinant ß3Gn-T2 and ß3Gn-T8 were mixed, the Vmax/Km value of the mixture was 9.3- and 160-fold higher than those of individual ß3Gn-T2 and -T8, respectively. Sephacryl S-300 gel filtration of the enzymes revealed that apparent molecular weights of each ß3Gn-T2, ß3Gn-T8, and the mixture were 90–160, 45–65, and 110–210 kDa, respectively, suggesting that ß3Gn-T2 and -T8 can form a complex with enhanced enzymatic activity. This is the first report demonstrating that in vitro mixed glycosyltransferases show enhanced enzymatic activity through the formation of a heterocomplex. These results suggested that ß3Gn-T8 and ß3Gn-T2 are cooperatively involved in the elongation of specific branch structures of multiantennary N-glycans.
Key words: N-acetylglucosaminyltransferase / enzyme complex / galactose / Pichia pastoris / tetraantennary N-glycan
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